The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) has been shown previously to mimic X-irradiation in altering cell cycle parameters in Hela cells [Kinzel, V., Richards, J., and Stöhr, M., Science (Wash. DC), 210: 429-431, 1980]. These changes include a delay in G2 phase from which cells recover in the presence of TPA, which suggests an involvement of cellular mediators. In order to obtain information on the onset and the duration of the G2 delay, as well as the onset and rate of recovery, a time-lapse study has been carried out. The analysis of cells in prophase shows that at 10(-6) M and 10(-7) M concentrations, TPA and 12-O-retinoylphorbol-13-acetate (RPA) cause a G2 delay which lasts on the order of 3.5 to 4 h. Below 10(-7) M of RPA and below 10(-8) M of TPA a clear-cut inhibition of HeLa cells in G2 is no longer detectable by this method. These results for a given phorbol ester are dose dependent within a certain range but unlike the case of X-rays are not proportional to dose. Within the dose range studied the recovery rate follows the opposite order. At 10(-6) M TPA and RPA an indication of a parasynchronous burst is observed. At smaller concentrations or with less biological activity of phorbol ester, the cell multiplication rate approaches that of the control or remains even smaller. Possible reasons are discussed. The determination of the transition points seems to indicate that the cellular events inhibited in G2 occur shortly before visible prophase.